Immune responses occur in diverse anatomical sites, including protective responses to pathogens and dysregulated immune responses in autoimmune and inflammatory diseases. However, in humans, our knowledge of T cell differentiation and maintenance in tissues remains sparse, and derives almost exclusively from studies of peripheral blood. My laboratory has carried out a unique whole body analysis of human T cells in lymphoid and mucosal tissues using tissue obtained from individual organ donors in collaboration with the New York Organ Donor Network (NYODN). Thus far, we have analyzed T cells from blood and eight different tissues including lymphoid tissues (spleen, peripheral and tissue-draining lymph nodes), and mucosal tissues (lung, small and large intestines) from over 24 research-consented, previously healthy organ donors. Our results reveal that the composition of naive, memory and terminal effector CD4 and CD8 subsets in blood, lymphoid and mucosal tissues is intrinsic to the tissue site and remarkably similar between diverse donors. Moreover, these subsets differentially express markers of activation and homeostasis in mucosal tissues, lymphoid tissues and blood, suggesting either in situ effects on T cell differentiation. Our central hypothesis is that compartmentalization of human T cells in tissues throughout the body depends on multiple mechanisms including the extent of replication, expression of specific T cell receptors (TCR), and tissue-specific factors. In the proposed research, we will 1. Evaluate the role of replication and turnover in tissue compartmentalization of T cell subsets, by examining whether different replication history and turnover is associated with residence in lymphoid versus mucosal sites; 2. Assess how TCR diversity and clonality is related to compartmentalization of T cell subsets in diverse anatomic sites using cutting edge deep sequencing approaches to directly probe whether tissue sites are enriched for specific T cell clones or contain limited TCR diversity compared to blood; and 3. Define the tissue-specific influences on T cells in circulating compared to lymphoid and mucosal sites through transcriptome analysis, by applying RNAseq analysis of T cell subsets in different sites to obtain novel tissue-specific gene signatures. Results from the proposed studies will provide valuable, new insights to how human immune responses are generated, maintained and regulated, essential for the development of new strategies for targeting tissue-specific immune responses in protective immunity, transplantation and autoimmunity. RELEVANCE (See instructions): Translating findings ih basic immunology from mouse to human is limited by our lack of fundamental knowledge of human T cells in tissue sites where they direct immune responses and are maintained. The proposed research will investigate how human T cells are compartmentalized on the cellular and molecular level in lymphoid and mucosal tissue sites using a unique tissue resource from organ donors. The results obtained will lead to new strategies for tissue targeting of T cell responses in vaccines and immunotherapies.